1. Introduction
Status Epilepticus (SE) is a life-threatening medical condition often associated with persistent seizures and is recurrent by nature (Bassin, Smith, & Bleck, 2002; Brophy et al., 2012; Meierkord, Boon, & Engelsen, 2010). It accounts for about 3% to 5% of all cases of emergency admissions for seizure disorders. It also occurs in 2% to 16% of individuals with epileptic disorders (Hauser, 1990). The incidence of SE was estimated as 41 to 61 cases per 100000 patients per year (Bronstein et al., 2010; DeLorenzo, Towne, Pellock, & Ko, 1992).
Isoniazid, an anti-tuberculosis drug, induces SE by depleting brain level of Gamma-Aminobutyric Acid (GABA), a major inhibitory transmitter substance in the mammalian brain, through inhibition of pyridoxal-5-phosphate-dependent Glutamic Acid Decarboxylase (GAD) (Corda, Costa, & Guidtti, 1982; Uzman et al., 2013). Pyridoxal-5-phosphate is the active form of pyridoxine, a cofactor for GAD, and an enzyme required for GABA synthesis (Bassin et al., 2002; Brophy et al., 2012; Meierkord et al., 2010; Corda et al., 1982; Uzman et al., 2013). The decrease in GABA levels results in recurrent seizures that characterized SE (Corda et al., 1982; Uzman et al., 2013). Although isoniazid-induced seizure is known to respond poorly to currently available anticonvulsant drugs, intravenous diazepam is still used to control the seizure episodes in the absence of pyridoxine (Corda et al., 1982; Uzman et al., 2013; Tajender & Saluja, 2006; Romero & Kuczler, 1998). 
On this basis, diazepam and pyridoxine, serving as reference drugs, were compared with the current study test substance. However, pyridoxine is reported as the only effective antidote for isoniazid toxicity and should be given in doses equivalent to the amounts of the ingested isoniazid in order to be effective (Uzman et al., 2013; Tajender & Saluja, 2006; Romero & Kuczler, 1998). However, when the quantity of the ingested isoniazid is unknown, intravenous administration of an initial dose of 5 g of pyridoxine is recommended in literature (Uzman et al., 2013; Tajender & Saluja, 2006). Although intravenous pyridoxine is relatively inexpensive, the immediate its sufficient amount is not always available in emergency (Tajender & Saluja, 2006; Romero & Kuczler, 1998). Thus, new compounds should be found that can serve as alternatives for isoniazid.
On this basis, diazepam and pyridoxine, serving as reference drugs, were compared with the current study test substance. However, pyridoxine is reported as the only effective antidote for isoniazid toxicity and should be given in doses equivalent to the amounts of the ingested isoniazid in order to be effective (Uzman et al., 2013; Tajender & Saluja, 2006; Romero & Kuczler, 1998). However, when the quantity of the ingested isoniazid is unknown, intravenous administration of an initial dose of 5 g of pyridoxine is recommended in literature (Uzman et al., 2013; Tajender & Saluja, 2006). Although intravenous pyridoxine is relatively inexpensive, the immediate its sufficient amount is not always available in emergency (Tajender & Saluja, 2006; Romero & Kuczler, 1998). Thus, new compounds should be found that can serve as alternatives for isoniazid.
It also depletes endogenous antioxidant status of the cells thereby making the body organs more prone to the deleterious effect of oxidative stress (Adebayo et al., 2012; Ahadpour et al., 2016; Cevik et al., 2012). However, the brain is more susceptible to oxidative stress damage due to high content of oxidizable fatty acids, high demand of oxygen, and low levels of antioxidant status (Moreira et al., 2008).
Thus, the combination of isoniazid with a potent antioxidant agent such as Jobelyn (JB) (Benson et al., 2013) may help reduce brain damage and thus prevent the manifestations of various neurological complications associated with isoniazid neurotoxicity. 
JB is a dietary supplement obtained from Sorghum bicolor L. (Gramineae family) that won international recognition for anemia, arthritis, and relief of stress (Benson et al., 2013; Okochi, Okpuzor, Okubena, & Awoyemi, 2003). The most active compounds in JB include apigenin, luteolin, and naringenin, which possess various biological activi­ties (Benson et al., 2013; Umukoro, Oluwole, Eduviere,  Adrian, & Ajayi, 2015). Previous investigations revealed that JB has potent antioxidant and anti-inflammatory properties (Benson et al., 2013; Umukoro et al., 2015). Moreover, JB has been listed as one of the herbal remedies used by the populace to manage febrile seizures (Oshikoya, Senbanjo, Njokanma, & Soipe, 2008). 
It was previously established that JB prolonged the latency to convulsions induced by pentylenetetrazole in mice (Umukoro, Omogbiya, & Eduviere, 2013), which suggests its possible efficacy in controlling convulsive episodes (Umukoro et al., 2013). However, the current study aimed at investigating the possible protective effect of JB against isoniazid-induced convulsion, altered oxidative stress, and Glutamate Decarboxylase (GAD) activity in mice.

2. Methods 
2.1. Laboratory Animals
Male Swiss mice (weighed 20-22 g) used in the study were purchased from the Central Animal House, University of Ibadan, Nigeria. They were housed in plastic perplex cages at room temperature, had free access to rodent pellet diet, and water. The experimental procedure was conducted in accordance with the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. 

2.2. Drugs and Chemicals 
JB (Forever Product Lagos, Nigeria), isoniazid (Hamex Medica Ltd., UK), diazepam (Swipha Pharma, UK), TriChloroacetic Acid- TCA (Burgoyne Burbidges & Co., Mumbai, India), Thiobarbituric Acid (TBA) (Sigma, Germany), 5,5′-Dithio-Bis(2-Nitrobenzoic acid) (DTNB) (Sigma Aldrich, Germany), Glutamate (Sigma Aldrich, Germany), and Pyridoxine (Pauco Pharamceutical Industry, Nigeria) were used in the study. JB and other drugs were dissolved in distilled water immediately before use. The doses of JB were selected based on previous studies (Umukoro et al., 2013).

2.3. Effect of Jobelyn on the onset of seizure
The effect of JB on isoniazid-induced convulsion was assessed as described (Corda et al., 1982). The animals were divided into different treatment groups (n=6) and were given distilled water (control, 10 mL/kg), diazepam (5 mg/kg), pyridoxine (300 mg/kg) or JB (10, 25, 50 mg/kg) orally. Thirty minutes later, the mice were given isoniazid (300 mg/kg, IP) and were observed for two hours for latency to convulsion and death. 

2.4. Biochemical studies
New set of animals were pretreated orally with distilled water (control, 10 mL/kg), JB (10, 25, 50 mg/kg), diazepam (5 mg/kg) or pyridoxine (300 mg/kg) 30 minutes prior to intraperitoneal injection of isoniazid (300 mg/kg). Thereafter, the mice were euthanized 30 minutes  post-isoniazid injection through cervical dislocation under ether anesthesia. The brains were removed, weighed, and kept in 10% w/v phosphate buffer (0.1M; pH 7.4). The whole brains were then homogenized in 10% w/v phosphate buffer and the supernatants were used for the biochemical studies. 

 2.4.1. Determination of glutathione concentration
The brain level of reduced GSH was determined as described (Moron, Depierre, & Mannervik, 1979). Briefly, the supernatant (0.4 mL) of the brain tissue was added to 20% trichloroacetic acid (0.4 mL) and then, centrifuged at 10000 rpm for 20 min at 4°C. This mixture (0.25 mL) was added to 2 mL of 0.6 mM DTNB and the final volume was made up to 3 mL with phosphate buffer (0.2M, pH 8.0). Next, the absorbance was read at 412 nm against blank reagent using a spectrophotometer. The brain levels of reduced GSH were expressed as μM/g tissue.

2.4.2. Estimation of brain concentration of malondialdehyde
MDA concentrations in the brain supernatants were determined as previously described (Ohkawa, Ohishi, & Yagi, 1979). Briefly, distilled water (0.5 mL) and 10% TCA (1.0 mL) were added to 0.5 mL of each homogenate of the brain tissue. This mixture was then centrifuged at 3000 rpm for 10 minutes and 0.1 mL of thiobarbituric acid (0.375%) was added to 0.4 mL of the supernatant. This mixture was then incubated in a water bath at 80°C for 40 minutes. After cooling, the absorbance of the supernatant was read at 532 nm using a spectrophotometer. The brain levels of MDA were expressed as μM/g tissue.

2.4.3. Determination of GAD activity
Brain GAD activity was determined as earlier described (Cozzani, 1970). The brain supernatant (1 mL) was adjusted to pH 7 and then incubated at 37°C for a period of 5 min. Thereafter, the reaction started by adding 100 μL glutamate (10 mM) and decarboxylation of glutamate was measured at 340 nm against a blank including all the components except glutamate using spectrophotometer (Cozzani, 1970). 

2.5. Statistical analysis
The obtained data were analyzed with Graph Pad Prism software, version 4.0 and expressed as Mean±standard Error of the Mean (SEM). Statistical analysis was conducted using 1-way ANOVA followed by the Newman-Keuls post-hoc test. P values less than 0.05 were considered statistically significant.

3. Results
3.1 Jobelyn prolongs latency to seizure
As shown in Table 1, intraperitoneal injection of isoniazid (300 mg/kg) produced 100% convulsion and death, as all the animals convulsed and died within the experimental period. However, JB (50 mg/kg, PO) prolonged the latency to seizure episodes (P<0.05), but failed to prevent occurrence of seizure episodes or death (Table 1). In contrast, diazepam but not pyridoxine offered 30% protection against convulsions and death in isoniazid-treated mice (Table 1). 

3.2. Jobelyn reduces isoniazid-induced oxidative stress in mouse brain
Figures 1 A and B showed the effect of JB on the brain MDA and GSH levels in isoniazid-treated mice. Isoniazid (300 mg/kg, IP) altered the concentrations of these biomarkers of oxidative stress relative to those of the control (P<0.05). As presented in Figure 1 A and B, JB (10, 25, 50 mg/kg, PO) or diazepam (5 mg/kg, PO) attenuated the altered MDA and GSH levels in the brains of mice treated with isoniazid (P˂0.05). 

3.3. Effect of Jobelyn on GAD activity in mice brains
The effect of JB on GAD activity in mice with isoniazid-induced seizure is shown in Figure 2. JB (50 mg/kg, PO), but not diazepam or pyridoxine, significantly elevated the activity of GAD in isoniazid-treated mice. 
 

 

4. Discussion
The results of the current study revealed that JB did not prevent the occurrence of seizures induced by isoniazid in mice. However, the highest dose of JB significantly delayed the onset of seizures. In contrast, diazepam, but not pyridoxine, offered 30% protection against convulsions and death in isoniazid-treated mice. Moreover, JB and diazepam decreased the brain MDA level and increased the GSH content suggesting antioxidant properties. However, JB, but not diazepam or pyridoxine, produced a significant increase in GAD activity, which suggested its ability to elevate GABA levels in the brains of mice treated with isoniazid.
SE is one of the key features of isoniazid poisoning or overdose and refractory to conventional anticonvulsant drugs (Bassin et al., 2002; Brophy et al., 2012; Meierkord et al., 2010). SE induced by isoniazid is related to inhibition of GAD, an enzyme required for GABA synthesis (Bassin et al., 2002; Bronstein et al., 2010; Corda et al., 1982; Cevik et al., 2012). Thus, the depletion of brain GABA levels is associated with continuous seizures observed in animals exposed to high doses of isoniazid. Treatment approach for isoniazid toxicity involves intravenous injection of pyridoxine, an essential cofactor required for GABA synthesis (Bassin et al., 2002; Brophy et al., 2012; Meierkord et al., 2010).
However, the uncertainty of the amount of INH ingested and its immediate availability are often the major limitations to the usefulness of pyridoxine in managing isoniazid toxicity in clinical settings (Minns, Ghafouri, & Clark, 2010). In the current study, the reasons why pyridoxine unlike diazepam could neither prevent nor delay INH-induced convulsions in mice were not apparent in the current investigation except that previous studies showed that pyridoxine was ineffective against isoniazid-induced seizures in mice (Bonner, Peterson, & Weir, 1999). Moreover, the reason (s) of pyridoxine protection of rats but not mice against convulsions induced by isoniazid was not also obvious from the study of Bonner et al. (1999).
Previous studies show that anticonvulsant effect of a novel agent does not absolutely depend on the prevention of convulsions, but on its ability to prolong the latency to seizures (Kendall, Fox, & Enna, 1981). Moreover, compounds that only delay the latency to convulsions block the spread of seizures in an epileptic brain (Corda et al., 1982). Thus, the finding that JB prolonged the latency to isoniazid-induced convulsions suggests anticonvulsant potentials in mice. The finding that JB also increased brain activity of GAD, an enzyme responsible for the synthesis of GABA, suggests its ability to elevate GABA levels in the brain. It is instructive to note that reduced brain GABA concentrations are implicated in the pathophysiology of convulsion (McNamara, 1994). Thus, the increase of GABA levels in the brain is one of the major therapeutic approaches in treating patients with seizures (McNamara, 1994). Nevertheless, more studies are necessary to establish the role of GAD in the anticonvulsant potential of JB in isoniazid-induced convulsions in mice.
Increased oxidative stress and subsequent manifestation of other neurological complications are associated with isoniazid toxicity (Cicek et al., 2005; Adebayo et al., 2012; Ahadpour et al., 2016; Cevik et al., 2012). Isoniazid-induced oxidative tissue damage is related to the formation of reactive oxygen species, which in turn initiate lipid peroxidation in most organs of the body, especially the liver and the brain (Cicek et al., 2005; Adebayo et al., 2012; Ahadpour et al., 2016; Cevik et al., 2012).

 

It also depletes endogenous antioxidant status of the cells, thereby exposing body organs to the deleterious effect of oxidative stress (Adebayo et al., 2012; Ahadpour et al., 2016; Cevik et al., 2012). However, the brain cells are more prone to deleterious effect of oxidative stress due to high content of oxidizable fatty acids, high oxygen demand, and low levels of antioxidant molecules (Moreira et al., 2008).
In recent years, a great number of natural products are sought from plants to mitigate oxidative stress-mediated pathologies (Mahmoud, Germoush & Soliman, 2014). For example, berberine, an alkaloid found in a variety of plants protects the liver against isoniazid-induced hepatotoxicity via inhibition of oxidative stress (Mahmoud et al., 2014). In addition, several phytochemicals demonstrate neuroprotection and are still being evaluated as potential therapeutic agents for neurological diseases (Natalie, Kelsey, Heather,  Wilkins, & Linseman, 2010). Moreover, previous preclinical investigations revealed that JB had antioxidant and neuroprotective properties in various experimental models (Benson et al., 2013; Oyinbo, Dare, Avwioro, & Igbigbi, 2015). 
Meanwhile, JB also demonstrated antioxidant property in the mentioned studies as it attenuated isoniazid-induced increase in brain MDA level, a well-known marker of oxidative stress implicated in lipid peroxidative tissue damage. This finding was in agreement with the reports of previous studies (Oyinbo et al., 2015) and further suggested that JB supplementation may protect the neurons against isoniazid-induced neurotoxicity. On the other hand, GSH content in brain significantly decreased in isoniazid-treated mice. Glutathione is a key endogenous antioxidant agent that protects cellular constituents against the harmful effects of oxidative stress by scavenging ROS (Franco, Schonveld, Pappa, & Panayiotidis 2007). However, pretreatment with JB inhibited depletion of GSH suggesting its ability to scavenge ROS generated by isoniazid in mice brains. Thus, the antioxidant effect exhibited by JB suggests its potential benefits to prevent neurological complications associated with isoniazid therapy.
The results of the current study suggested that increased GAD activity might contribute to the ability of JB to prolong the latency to convulsions induced by isoniazid. The current study also suggests that JB supplementation might be useful as a potential antioxidant agent in preventing oxidative stress-induced neurological complications associated with anti-tuberculosis therapy with isoniazid. 

Ethical Considerations
Compliance with ethical guidelines
The experimental procedures utilized in this study were in accordance with the National Institute of Health (NIH) guidelines for the care and use of Laboratory animals.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors contributions
The authors contributions is as follows: Solomon Umukoro, Stephen Asehinde and Osarume Omorogbe were involved in the conceptualization of the study and original draft preparation of the manuscript;  Abayomi Ajayi, Adewale Bakre, Stephen Asehinde and Adaeze Adebesin carried out the experimental studies and data analysis; and Solomon Umukoro, Abayomi Ajayi and Osarume Omorogbe wrote the final version of the manuscript.

Conflict of interest
The authors declare no conflict of interest.

Acknowledgements
The authors would like to thank the technical staff of the Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria for their kind assistance. We also thank professor O.G. Ademowo of the Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria for providing the glutamte for the assay of GAD. 


References